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Herb–drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay
Accepted Manuscript Title: Herb-drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay Author: Shiv Bahadur Pulok K Mukherjee Subrata Pandit SKMilan Ahmmed Amit Kar PII: DOI: Reference:
S2213-7130(16)30033-5 http://dx.doi.org/doi:10.1016/j.synres.2016.12.001 SYNRES 23
To appear in: Received date: Accepted date:
5-12-2016 5-12-2016
Please cite this article as: Shiv Bahadur, Pulok K Mukherjee, Subrata Pandit, SKMilan Ahmmed, Amit Kar, Herb-drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay, Synergy http://dx.doi.org/10.1016/j.synres.2016.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Herb-drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay Shiv Bahadur, Pulok K Mukherjee*, Subrata Pandit, SK Milan Ahmmed, Amit Kar School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
*Corresponding author Prof. Pulok K Mukherjee, PhD, FRSC Director, School of Natural Product Studies Jadavpur University, Kolkata 700032, Kolkata, India. Telefax: +91-33-24146046 E-mail: [email protected]
Graphical abstract
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Highlights
Standardization of Berberis aristata extract though RP-HPLC method
CYP450 inhibition assay by CYP-CO complex assay and florescence inhibition assay
Herb-drug interaction study of Berberis aristata and oral hypoglycaemic drugs
Combination effects of Berberis aristata, their phytoconstituent and oral hypoglycaemic drug
Abstract Herbal medicines have been used from ancient times for the management of several disease and ailments along with prescribed medicine without consulting the physicians. Berberis aristata DC. has been widely used in management of diabetes as alternative therapy in India and other Asian countries. An approach has been made to evaluate the possible cytochrome P450 (CYP450) enzyme inhibition potential with the bark of the anti-diabetic herb Berberis aristata DC. along with combination of oral hypoglycaemic agents like glimepiride and gliclazide. Bioactive compound was quantified through RP-HPLC, in order to standardize the plant extracts and interaction potential of standardized extract and bioactive compounds with oral hypoglycaemic drugs were evaluated. Herb-drug interaction potential of the test samples were evaluated by CYP450-carbonmonoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human CYP450 isoforms such as CYP3A4, CYP2D6, CYP2C9 and CYP1A2 isozymes were analyzed through fluorescence microplate screening assay. The combination index (CI) - isobologram method was applied for the prediction of combination effects. Plant extract showed significantly higher IC50 value than respective positive control against CYP3A4, 2D6, 2C9 and 1A2. Combination of the test extract and the phytoconstituent present therein along with oral hypoglycemic drugs also showed significantly less (P<0.001, P<0.01) interaction potential than known CYP450
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inhibitors. Present study concluded that selected medicinal plant unlikely to cause significant herb-drug interactions and safe to use in management of diabetes.
List of abbreviations: CYP450
Cytochrome P450
HTS
High - throughput screening
IC50
The 50% inhibitory concentration
RP-HPLC
Reverse phase high-performance liquid chromatography
CYP450-CO
Cytochrome P450- carbon monoxide
RLM
Rat liver microsome
CI
Combination Index
Keywords: Herb-drug interaction, RP-HPLC, Berberis aristata DC., Cytochrome P450, Berberine, Combination index
1. Introduction Berberis aristata DC. (Family: Berberidaceae) is traditionally used in the Indian Systems of Medicines (ISM) for several ailments. The bark of this plant posses anti-diabetic, anti-inflammatory, hypotensive, immuno-stimulating, wound healing preparations, and also prevents infection of eyes [1, 2]. Plant extracts of Berberis aristata are widely used in the management of diabetes in therapies of the Indian system of medicine. Several studied revealed that Berberis aristata has an anti-diabetic activity through different mechanisms such as mimicking insulin, increasing insulin action by activating AMPK (5′ adenosine monophosphateactivated protein kinase), reducing insulin resistance through protein kinase
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C-dependent up-regulation of insulin receptor expression, glycolysis induction, and by inhibiting DPP-IV activity [3]. Cytochrome P450 (CYP450) enzymes are major drug metabolizing enzymes and indicators of herb-drug interactions [4]. An inhibition or induction of CYP450 is the most common mechanism for the pharmacokinetic interactions of herb and drugs [5]. The safety issues, specifically herb–drug interaction data are important for the safe clinical practice of herbal medicines [6]. Multidrug combination therapy is common nowadays to achieve a synergistic therapeutic efficacy. Even though herbal medicines are gaining popularity as an alternative or adjuvant treatment globally, herb-drug and herb-herb interactions are a current topic of debate. Still effects of the herbal medicine in the combination of modern pharmaceuticals and possible adverse effects from herb - drug interactions remain to be verified. These studies are being suggested by most of the international regulatory bodies such as European Agency for Medicines (EMA) and US Food and Drug Administration (FDA) in the process of herbal drug development [7]. Herbal medicines having anti-diabetic activity are most commonly consumed as a dietary supplement with the prescribed medicines. Glimepiride and gliclazide are second generation sulphonylurea (oral hypoglycaemic drugs) most commonly used in the treatment of non-insulin-dependent diabetes mellitus [8]. The development of a marker profiling as a standardization parameter is of great importance for quality control management and batch to batch reproducibility of their therapeutic potency by the herbal drug manufacturers [9]. Based on these concepts the present study was designed to standardize the B. aristata extract with respect to berberine through reverse phase high-performance liquid chromatography (RPHPLC). The herb-drug interaction potential of these plant extracts, major phytoconstiuents in combination with oral hypoglycaemic drugs were investigated in order to obtain information about synergistic effects and about the safety profile in a combination therapy. Further we applied the combination index (CI)-isobologram method which is widely used to study
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interaction to determine the nature of toxicological interactions of the B. aristata extract and oral hypoglycaemic drug, it allows computerized quantitation of synergism, additive effect and antagonism.
2. Materials and methods 2.1. Chemicals Vivid® CYP450 Screening Kit (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) and specific Vivid® Substrates were purchased from Invitrogen Drug Discovery Solutions (USA). Vivid® blue screening kit included baculosome (isozymes + NADPH-P450 reductase); regeneration system (glucose-6-phosphate + glucose-6-phosphate dehydrogenase) and NADP+ were used for the study. Ketoconazole and α-naphthoflavone were obtained from Merck (Mumbai, India). Quinidine and sulfaphenazole were procured from Sigma (Steinheim, Germany). Fluorimetric screening was performed using the 96 well black microplate (NUNC, Roskilde, Denmark; Catalogue no.: 137101). Berberine, glimepiride and gliclazide were purchased from Sigma Aldrich (Steinheim, Germany). Milli-Q Water (Millipore, USA) was used throughout the analysis; HPLC grade methanol, acetic acid, and other analytical grade solvents were procured from Merck (Mumbai, India). 0.45 μm syringe filter and membrane filter were obtained from Phenomenex (Torrance, CA) and Millipore (Billerica, MA) respectively. Milli-Q Water (Millipore, USA) was used throughout the analysis. All the chemical and reagents used were freshly prepared and analytical grade.
2.2. Plant material and extraction Bark of Berberis aristata was procured from the local vender and authenticated by Dr. S. Rajan, Field Botanist, Tamilnadu. Voucher specimen (SNPS-JU/1047) has been deposited at the School of Natural of Product Studies, Jadavpur University, Kolkata, for the future
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reference. 200 gm collected plant material was extracted with 95% ethanol by cold maceration method. Solvent was removed by rotary vacuum evaporator (EYELA, Tokyo, Japan) in reduced pressure at 40°C, leaving semi-solid residue. The semisolid residue was dried under lyophilisation to get fine powder and yield of the extract was 12.38%.
2.3. Standardization of extracts through RP-HPLC The extract of B. aristata was standardized through RP-HPLC. The RP-HPLC (Waters 600, Milford, MA, USA) system was comprised of rheodyne-7725 injection valve with a sample loop (20 mL), vacuum degasser, quaternary pump and photo-diode array detector (PDA) with data acquisition by Empower TM 2 software. Different concentrations of the standard were prepared for the preparation of the calibration curve. Samples were filtered through 0.45 mm (NYL) syringe filter before injection. Retention time for all the eluting components were set within16 min run time. Chromatography was performed on a Spherisorb C18 column (250mm × 4.6 mm, 5 mm; Waters, Ireland) fitted with a C18 guard column (10 × 3.0 mm). The sample elution was performed at 25°C and detected at 254 nm. Calibration curve was constructed by the corresponding peak area and the concentration of standards by means of linear regression.
2.4. In vitro Cytochrome P450 inhibition studies 2.4.1. Preparation rat liver microsomes Rat liver microsomes were prepared based on the method described by Ponnusankar et al. (2011) [10]. In short, rats were sacrificed and the liver was isolated and treated with 1.15% potassium chloride solution (KCl) and then its size reduction was undertaken. 20% of the homogenized liver suspension was centrifuged through Beckman Coulter 64RALLEGRA at 9000 x g for 20 min. The clear supernatant was collected and subjected for re-
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centrifugation at 105,000 x g 4 1C for 1 h (ultra centrifuge- SORVALL RC100). From the centrifuged suspension pellet microsomal fractions were collected and re-suspended in 1.15% KCl solution and kept at - 80 ºC for next use. Then estimation of protein was performed by a modified biuret method using bovine serum albumin (BSA) as standard by an AMS Photoanalyzer PF-2 (Roma, Italy). All experiments were performed as approved by the Institutional Animal Ethical Committee with the ethical guidelines as provided by the committee (approval number: AEC/PHARM/1501/05/2015) for the purpose of control and supervision of experiment on animals (CPCSEA), India.
2.4.2 Cytochrome P450-carbon monoxide (CYP-CO) complex assay CYP450-CO complex assay was performed with pooled rat liver microsomes (RLM) in 96 well microplates by the method described by Ponnusankar et al., (2011) [11]. In brief, the plant extract was mixed with ethanol and DMSO. It was incubated with RLM (liquefied with phosphoglycerol buffer; pH 7.4). The reaction between CYP450 and extract was initiated by adding an NADPH generating system (4.20 mg/mL of NADP + in solution of 100 mM glucose 6-phosphate, 100 mM MgCl2 and 100 U/mL glucose 6-phosphate dehydrogenase). The reaction between enzyme and extract was carried out in two microplates: one under room temperature (P) and another in carbon monoxide (CO) chamber (PC) for 15 min, while 0.5 M sodium hydrosulphite was used as the reducing agent. Two aliquots were placed in microplate wells and the other one was designed as reduced P450 (P) and other one was reduced P450-CO complex (PC). The P was sealed and kept outside the chamber whereas the PC well was kept in CO chamber and incubated for 15 min. All the samples were reduced by addition of 0.5 M sodium hydrosulfite (SHS) fresh solution. Visible appearance of PC sample was yellow while sample P remained colourless. The difference in absorbance of samples at 450 and 490 nm was monitored using BIORAD microplate reader
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(Model 680XR). Ketoconazole was used as positive control and proper solvent controls were used for the study. Ethanol and DMSO were used as solvent for the cytochrome assay and had no interaction with spectral absorption while the solvent effect was neutralized by deionized water (Milli- Q water) and used as a solvent control. The result was expressed as percentage inhibition of CYP450. Concentration of CYP450 was calculated using the following formula [12]. [CYP450] (mM) = (ΔAPC-ΔAP)/91 Where, ΔAPC = absorbance difference of the PC sample and ΔAP = absorbance difference of the P sample. Percentage inhibition was calculated using the following formula: Percentage inhibition (PI) = (Blank - Test) × 100/Blank
2.4.3. Flurogenic high throughput screening (HTS) assay cytochrome P450 enzymes inhibition assays The assay was performed as per the method described by the Pandit et al., (2011a; 2012). In brief 145 μl NADPH-Co factor mixture (1.3 mM NADP+, 66mM MgCl2 and 66 mM glucose 6-phosphate) was added to each well of the first row of black 96 micro-well plates. Then 100 μl cofactor mixtures were added to the remaining wells except the blanks. 5 μl of extract solution was added to the first row of each well. Two-fold serial dilution was made and plates were incubated for 10 min at 37 °C. Quinidine (1 μM), ketoconazole (10 μM), sulfaphenazole (10 μM) and α-naphthoflavone (3 μM) were used as positive inhibitors for CYP2D6, CYP3A4, CYP2C9 and CYP1A2 respectively. 100 μl enzyme-substrate solutions were prepared in phosphate buffer and were used for starting the reaction and kept for 30 min. After incubation the reaction was stopped by addition of 0.5 M tris base. Product formation from the fluorogenic probes were determined from the fluorescence data at eight different concentrations of the inhibitors and the test. All measurements were performed in
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triplicate. Calculation of IC50 values was based on the curves of mean enzyme activity versus inhibitor concentration [12]. The data were analyzed and result was expressed as IC50 value of the extract. Percentage inhibition = 100 – [(Signal of well-Blank)/ (Solvent control-Blank) × 100] IC50 = [{(50-LP) × (HC-LC) + LC}/ {HP-LP}] Where, LP = Low percentage of inhibition; HP = High percentage of inhibition; LC = Low concentration; HC = High concentration 2.5. Combination index (CI) - isobologram for determination of herb-drug interaction The herb drug interactions were investigated by flourescence microplate screening assays using 96-well micorplate readers. The IC-50 values were determined by different concentrations of each sample in triplicate. Combination index (CI) - isobologram equations was developed for the determination of the combined effect of herb-drug interaction. Two oral hypoglycaemic drugs were selected with the different combinations of plant extract and their active metabolite such as Berberis aristata + gliclazide, berberine +glimepiride, berberine + gliclazide and Berberis aristata + glimepiride for all the selected isozymes. The computer software CompuSyn [13] was used for the determination of the combination index (CI) to find out the combination effect. CI < 1, CI=1 and CI > 1 indicates synergism, concentration addition and antagonism respectively [14]. The CI method allows quantitative determination of synergism or antagonism and the elucidation of the mechanism of combined actions needs to be explored in other ways [15].
2.6. Statistical analysis Results were presented as Mean ± SEM. The results were subjected to one-way analysis of variance (ANOVA) followed by the Bonferroni test. The difference between the
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means was considered significant when p<0.05 and above. The statistical analysis was performed using the GraphPad InStat Version 5 (USA).
3. Results 3.1. Standardization of extract through RP-HPLC The extract was standardized by RP-HPLC under isocratic conditions using the bexternal standard calibration technique. The Bioactive compound was identified by comparing with the respective retention time of the reference standard. Calibration curve was plotted by plotting peak areas against concentrations at five standard marker ranges from 100 – 500 μg/ml for barbarine with the flow rate of 1mL/min. Standard compound showed good linearity between concentrations and the peak area. The correlation coefficient (r2) of berberine was found to be 0.998. The chromatograms obtained from RP-HPLC analyses of the B. aristata extract and biomarkers have been shown in Fig.1. Optimum separation of berberine was achieved by using methanol: water with 1% acetic acid with 60:40 ratios (v/v). Retention time of berberine was found to be 14.28 min. Results were considered satisfactory and acceptable for subsequent quantitative analysis. The percentage of standard berberine was found to be 2.12% (w/w). 3.2. Cytochrome inhibition study 3.2.1. Cytochrome P450-CO complex assay CYP-CO assay was performed to evaluate the inhibitory potential of B. aristata and bioactive compounds in combination with glimepiride and/or gliclazide (oral hypoglycaemic drugs). The concentration of protein present in isolated rat liver microsomes was found to be 6.70 mg/ml. The CYP450 concentration of RLM was found to be 0.618 nmol/mg protein. The study was performed with different concentrations of the test solutions and the change in the absorption spectra was measured. The percentage inhibition of CYP450 was calculated and the comparison of percentage of inhibition of the individual test substances with respect 10
to positive control has been shown in Fig. 2. To ensure the possible inhibition by the extract, suitable solvent control was used in this experiment and the percentage inhibition was calculated after nullifying the solvent effect. Result indicated the concentration-dependent inhibition of CYP450. Positive inhibitor ketoconazole showed significantly higher (p<0.001) inhibition potential than extracts, bioactive, oral hypoglycaemic drugs and their combinations. Results showed the interaction of extracts with the pooled CYP450 was higher than compared to their designated bioactive molecule. This effect may be related to potentially synergistic activities of some other active molecules present in the extract [16, 17].
3.2.2. Fluorogenic HTS assays For the characterization of the herb - drug interactions potential, CYP isozymes specific high-throughput screening (HTS) assays is an important tool. The B. aristata extract and major bioactive principle berberine were evaluated for their capability to predict CYP450 mediated adverse drug effects of oral hypoglycaemic drugs such as glimepiride and gliclazide reaction when administered concomitantly. The interaction was studied with different CYP isozymes like CYP3A4, 2D6, 2C9 and 1A2 for their potential activity. The assay was based on the ability of a drug to compete with different fluorogenic substrates for different CYP isoforms. Coumarin derivatives were used as the probe substrates, which generated fluorescent products after dealkylation by CYPs. BOMCC (7-benzyloxymethyloxy- 3cyanocoumarin) was used as substrate for CYP3A4 and CYP2C9. In case of CYP2D6 and CYP1A2, EOMCC (7-ethoxymethoxy- 3-cyanocoumarin) was used as substrate. 7Hydroxycoumarin and 7-hydroxy-3-cyanocoumarin by products were formed against BOMCC and EOMCC after the enzymatic reaction. Positive controls for each isozyme were
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used to confirm the assays precision. Testing of pure compounds indicated the overall activity explainable by tested compounds or caused by other constituents in the extract. Test samples showed concentration dependent inhibitory activity on CYP1A2, 2C9, 2D6 and 3A4 isozymes (Fig. 3). All the samples were assayed in triplicate and IC50 values were determined. IC50 values of the Berberis aristata extract, berberine and their combinations with oral hypoglycaemic drugs have been represented in Table 1. The results revealed that extract and its bioactive compound have less inhibition potential on the tested isozymes compared to their respective positive inhibitors. The study indicated that bioactive constituents have higher inhibition potential compared to extracts. Higher enzyme inhibition potential by the extracts may be due to the synergistic effects of other constituents present in the extract. More interestingly berberine + glimapiride and berberine + gliclazide combinations showed more inhibition potential against the isozymes studied. Berberine showed significantly less interaction potential against CYP450 isozymes compared to respective positive inhibitors. Test substances produced only minor inhibition of CYP1A2, 2C9, 2D6 and 3A4 isozymes. The cocktail of phyto-constituent and drugs showed significantly higher IC50 value compared to positive inhibitors against CYP1A2, 2C9, 2D6 and 3A4 isozymes. Results indicated that test substances have significantly less potential to interact with co-administered drug. 3.3. Combination index (CI) – isobologram for her-drug interaction The combination index (CI) method can be used for the prediction of combination action on all effect levels for multiple components and is widely used [18]. Herb - drug interaction of the Barberis aristata extract and their bioactive molecule berberine with the oral hypoglycaemic drugs glimepiride and gliclazide were evaluated. On the basis of observed CI values the binary mixture showed slightly synergetic effects of the oral hypoglycaemic drug with the Barberis aristata extract and their bioactive compound berberine (Table 2).
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4. Discussion Development of marker profiling for standardization of herbal medicine is one of the most important parameter for maintaining their quality and to enrich the knowledge about the optimal concentrations of bio-active constituents present therein [19]. During herbal drug development the herb-drug interactions and nutritional supplement-drug interactions should not be ignored. Most of herbal medicines have been used without the prior consideration of physicians and there are chances of synergetic effects [20]. Two or more drugs administered together are considered to act synergistically if the observed effect is greater than the effect expected from the effect of each drug administered alone [21]. Considering the worldwide popularity of herbal medicine with the development of several herbal preparations, incidence of herb-drug interactions is predicted to be increased [22]. An approach has been made to evaluate the synergetic effects of anti-diabetic Indian medicinal plant B. aristata through CYP-CO complex method and high throughput fluorescence screening assays. Orally administered medicinal products are mostly metabolized by gut flora before being absorbed into the systematic circulation [23]. CYP450 has very significant role as metabolizing enzyme which is involved in the biotransformation of substances taken by oral route while the pre- systemic metabolism of these drugs takes place before entering into liver cell [17]. The interaction between herb and drug with CYP450 enzyme may alter plasma concentrations and may lead to toxic effects. Fluorescence based CYP450 screening method for the herbal medicine is important to obtain the knowledge about herb-drug interaction. Concentration dependent CYP450 inhibition was found in fluorescence screening assay by the test and standard [24]. CYP3A4, 2C9, 2D6 and 1A2 are clinically most important drug metabolizing enzymes. Although B. aristata showed little inhibition potential against CYP3A4 isozyme but with combination of oral hypoglycaemic drugs there was negligible inhibition. This effect may be due to the
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glimepiride which is metabolized by CYP2C9 and has no effect with CYP3A4. Gliclazide mainly metabolized by CYP2C9 and 2C19 isozymes [8]. Results showed that B. aristata extract and the combination of oral hypoglycaemic drugs have not significant drug interaction potential against CYP2C9 & 3A4. In other word B. aristata and their bioactive compound did not involve in interaction with other drug metabolism. Combination index value showed that the B. aristata has little synergetic effects in the combination of the oral hypoglycaemic drugs. This may be due to the Barberis aristata and berberine also have hypoglycemic activity [25]. Further in-vivo and clinical study need to be performed in future for a better understanding of herb-drug interaction. 5. Conclusion With the increasing demand and popularization "herbs" and food as medicine across the globe, the quality and safety assessment of raw material becomes an essential parameter before marketing. The observed results by CYP-CO complex method and fluorogenic assay suggested that the B. aristata extract and their bioactive compounds in combination with oral hypoglycaemic agents, glimepiride and gliclazide have significantly less inhibitory effect on drug metabolising enzymes. Test substances were less likely to produce significant herb-drug interactions when consumed along with conventional therapeutics.
Conflict of interest Authors have no conflict of interest.
Acknowledgement Authors would like to express their gratitude to the Department of Biotechnology (DBT), Government of India, New Delhi, for providing TATA Innovation Fellowship to Dr. Pulok K. Mukherjee, School of Natural Product Studies, Jadavpur University, Kolkata. 14
References 1.
H.S. Yogesh, V.M. Chandrashekhar, H.R. Katti, S. Ganapaty, H.L. Raghavendra, G.K. Gowda, Anti-osteoporotic activity of aqueous-methanol extract of Berberis aristata in ovariectomized rats, J. Ethnopharmacol. 134 (2011) 334–338.
2.
D. Potdar, R.R. Hirwani, S. Dhulap, Phyto-chemical and pharmacological applications of Berberis aristata, Fitoterapia 83 (2012) 817–830.
3.
J. Singh, P. Kakkar, Antihyperglycemic and antioxidant effect of Berberis aristata root extract and its role in regulating carbohydrate metabolism in diabetic rats, J Ethnopharmacol 123 (2009) 2-6.
4.
H. Kim, H.K. Choi, T.C. Jeong, Y. Jahng, D.H. Kim, S. Lee, S. Lee, Selective inhibitory effects of mollugin on CYP1A2 in human liver microsomes, Food Chem. Toxicol. 51 (2013) 33-37.
5.
S. Pandit, P.K. Mukharjee, K. Mukharjee, R. Gajbhiye, M. Venkatesh, S. Ponnusankar, S. Bhadra, Cytochrome P450 inhibitory potential of selected Indian spices - possible food drug interaction, Food Res. Int. 45 (2012) 69–74.
6.
E.J. Choi, J.B. Park, K.D. Yoon, S.K. Bae, Evaluation of the in vitro/in vivo potential of five berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) commonly used as herbal supplements to inhibit uridine diphospho-glucuronosyltransferase, Food Chem. Toxicol. 72 (2014) 13-19.
7.
B. Kamble, A. Gupta, I. Moothedath, L. Khatal, S. Janrao, A. Jadhav, B. Duraiswamy, Effects of Gymnema sylvestre extract on the pharmacokinetics and pharmacodynamics of glimepiride in streptozotocin induced diabetic rats, Chem. Bio. Inter. 245 (2016) 30-38.
15
8.
A. Tornio, M. Niemi, P.J. Neuvonen, T.B. Janne, Drug interactions with oral antidiabetic agents: pharmacokinetic mechanisms and clinical implications, Trends in Pharmacol. Sci. 33 (2012) 312-322.
9.
P.K. Mukherjee, S. Bahadur, S.K. Chaudhary, A. Kar, K. Mukherjee, Quality Related Safety Issue-Evidence-Based Validation of Herbal Medicine Farm to Pharma in P.K. Mukherjee (Edt) Evidence based validation of herbal medicine, Netherlands, USA 2015, 1-28.
10. S. Ponnusankar, S. Pandit, , R. Babu, A. Bandyopadhyay, P.K. Mukherjee, Cytochrome P450 inhibitory potential of Triphala- A Rasayana from Ayurveda. J. Ethnopharmacol. 133 (2011) 120-125. 11. S. Ponnusankar, S. Pandit, M. Venkatesh, A. Bandhopadhyay, P.K. Mukherjee, Cytochrome P450 inhibition assay for standardized extract of Terminalia chebula Retz. Phytother. Res. 25 (2011) 151- 154. 12. S. Pandit, S. Ponnusankar, A. Bandyopadhyay, S. Ota, P.K. Mukherjee, Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6, Phytother. Res. 25 (2011) 1429- 34. 13. T.C. Chou, N. Martin, 2005,CompuSyn for drug combinations: PC software and user's guide: A computer program for quantification of synergism and antagonism in drug combinations and the determination of IC50 and ED50 and LD50 values. ComboSyn, Inc., Paramus, NJ. 14. J. Fu, N. Zhang, J.H. Chou, H. Dong, S. Lin, G.S. Ulrich-Merzenich, T. Chou, Drug combination in vivo using combination index method: Taxotere and T607 against colon carcinoma HCT-116 xenograft tumor in nude mice, Synergy 3 (2016) 15-30. 15. I. Rodea-Palomares, A.L. Petre, K. Boltes, F. Legans, J.A. Perdigón-Melón, R. Rosal, F. Fernández-Piñas, Application of the combination index (CI) - isobologram equation to
16
study the toxicological interactions of lipid regulators in two aquatic bioluminescent organisms, Water Res. 44 (2010) 427-438. 16. S. Pandit, P.K. Mukherjee, S. Ponnusankar, M. Venkatesh, N. Srikanth, Metabolism mediated interaction of α-asarone and Acorus calamus with CYP3A4 and CYP2D6, Fitoterapia 82 (2011) 369-74. 17. R.K. Harwansh, K. Mukherjee, S. Bhadra, A. Kar, S. Bahadur, A. Mitra, P.K. Mukherjee, Cytochrome P450 inhibitory potential and RP-HPLC standardization of Trikatu - A Rasayana from Indian Ayurveda, J. Ethnopharmacol. 153 (2014) 674-681. 18. Y. Wang, C. Chen, Y. Qian, Z. Zhao, Q. Wang, X. Kong, Toxicity of mixtures of λcyhalothrin, imidacloprid and cadmium on the earthworm Eisenia fetida by combination index (CI) - isobologram method, Ecotoxicol Environment Safety. 111 (2015) 242-247. 19. P.J. Houghton and P.K. Mukherjee, Evaluation of herbal medicinal products, 1st ed. Pharmaceutical Press, London. 2009. 20. G.S. Ulrich-Merzenich, Combination screening of synthetic drugs and plant derived natural products-Potential and challenges for drug development, Synergy 1 (2014) 59-69. 21. N.J. Sucher, Searching for synergy in silico, in vitro and in vivo, Synergy 1 (2014) 3043. 22. S. J. Brantley, A.A. Argikar, Y. S. Lin, S. Nagar, M.F. Paine, Herb-drug interactions: Challenges and opportunities for improved predictions. Drug Metab. Dispos. 42 (2014) 301-317. 23. S.M. Ahmmed, P.K. Mukherjee, S. Bahadur, A. Kar, K. Mukherjee, S. Karmakar, A. Bandyopadhyay, Interaction potential of Trigonella foenum graceum through cytochrome P450 mediated inhibition. Ind. J. Pharmacol. 47 (2015) 530-534.
17
24. P.K. Mukherjee, S. Ponnusankar, S. Pandit, P.K. Hazam, M. Ahmmad, K. Mukharjee, Botanicals as medicinal food and their effects on drug metabolizing enzymes, Food Chem. Toxicol. 49 (2011) 3142–3153. 25. Y. Wang, T. Campbell, B. Perry, C. Beaurepaire, L. Qin, Hypoglycemic and insulinsensitizing effects of berberine in high-fat diet- and streptozotocin - induced diabetic rats, Metabolism, 60 (2011), 298-305.
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Figure legends:
Fig.1: RP-HPLC chromatograms of berberine (A), B. aristata extract (B)
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Fig.2: Inhibitory effects of test extract, bioactive compounds and oral hypoglycaemic drugs and their combinations on pooled rat liver microsomes, (Values are expressed as mean ± SEM (n=3); **p<0.001 and ***p<0.01)
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Fig 3: Concentration dependant percentage inhibition of Berberis aristata extract, berberine, oral hypoglycaemic drugs and their combinations on drug metabolizing isozymes CYP3A4 (A), CYP2D6 (B), CYP2C9 (C) and CYP1A2 (D): (Values are in Mean ± SEM)
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Table 1: IC50 value (µg/ml) of extract and combination of extract and hypoglycemic drug: (One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test) Test samples
CYP3A4
CYP2D6
CYP2C9
CYP1A2
Berberis aristata
138.88±1.93
169.13±11.03
146.57±5.66
181.27±5.97
Berberis aristata + Glimepiride Berberis aristata + Gliclazide Berberine
81.88±2.14
110.94±2.60
98.32±2.04
131.16±4.12
96.65±2.27
105.01±1.22
83.95±1.57
125.44±2.75
65.80±3.63
85.60±2.57
79.85±5.23
98.64±5.70
Berberine + Glimepiride Berberine + Gliclazide
49.38±4.44
78.00±137
86.64±0.98
92.73±1.92
40.08±2.35
81.53±2.24
74.95±1.90
94.10±1.28
Glimepiride
56.82±5.93
40.48±5.21
46.32±5.79
79.59±3.29
Gliclazide
43.28±6.16
50.50±5.59
36.38±4.12
63.06±2.35
Positive control
Ketoconazole
Quinidine
Sulfaphenazole
α-naphthoflavone
0.05 ± 1.24
0.64 ± 0.94
0.09 ± 1.92
0.39 ± 0.58
22
Table 2: Combination index (CI) values of Berberis aristata and berberine in the binary combinations with glimepiride and gliclazide
ED50
CI values ED75 ED90
ED95
CYP3A4 Berberis aristata + Glimepiride
0.88
0.79
0.71
0.66
Berberis aristata + Gliclazide
0.82
0.85
0.89
0.91
Berberine + Glimepiride
0.92
0.85
0.78
0.74
Berberine + Gliclazide
0.86
0.80
0.75
0.71
Berberis aristata + Glimepiride
0.80
0.71
0.77
0.87
Berberis aristata + Gliclazide
0.96
0.92
0.91
0.93
Berberine + Glimepiride
0.92
0.85
0.80
0.77
Berberine + Gliclazide
0.85
0.72
0.75
0.85
Berberis aristata + Glimepiride
0.83
0.58
0.59
0.65
Berberis aristata + Gliclazide
0.98
0.72
0.79
0.93
Berberine + Glimepiride
0.94
0.81
0.70
0.63
Berberine + Gliclazide
0.87
0.83
0.80
0.77
Berberis aristata + Glimepiride
0.69
0.66
0.82
1.00
Berberis aristata + Gliclazide
0.78
0.60
0.67
0.78
Berberine + Glimepiride
0.92
0.91
0.91
0.91
Berberine + Gliclazide
0.81
0.77
0.73
0.71
Test samples
CYP2D6
CYP2C9
CYP1A2
23
S2213-7130(16)30033-5 http://dx.doi.org/doi:10.1016/j.synres.2016.12.001 SYNRES 23
To appear in: Received date: Accepted date:
5-12-2016 5-12-2016
Please cite this article as: Shiv Bahadur, Pulok K Mukherjee, Subrata Pandit, SKMilan Ahmmed, Amit Kar, Herb-drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay, Synergy http://dx.doi.org/10.1016/j.synres.2016.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Herb-drug interaction potential of Berberis aristata through cytochrome P450 inhibition assay Shiv Bahadur, Pulok K Mukherjee*, Subrata Pandit, SK Milan Ahmmed, Amit Kar School of Natural Product Studies, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India
*Corresponding author Prof. Pulok K Mukherjee, PhD, FRSC Director, School of Natural Product Studies Jadavpur University, Kolkata 700032, Kolkata, India. Telefax: +91-33-24146046 E-mail: [email protected]
Graphical abstract
1
Highlights
Standardization of Berberis aristata extract though RP-HPLC method
CYP450 inhibition assay by CYP-CO complex assay and florescence inhibition assay
Herb-drug interaction study of Berberis aristata and oral hypoglycaemic drugs
Combination effects of Berberis aristata, their phytoconstituent and oral hypoglycaemic drug
Abstract Herbal medicines have been used from ancient times for the management of several disease and ailments along with prescribed medicine without consulting the physicians. Berberis aristata DC. has been widely used in management of diabetes as alternative therapy in India and other Asian countries. An approach has been made to evaluate the possible cytochrome P450 (CYP450) enzyme inhibition potential with the bark of the anti-diabetic herb Berberis aristata DC. along with combination of oral hypoglycaemic agents like glimepiride and gliclazide. Bioactive compound was quantified through RP-HPLC, in order to standardize the plant extracts and interaction potential of standardized extract and bioactive compounds with oral hypoglycaemic drugs were evaluated. Herb-drug interaction potential of the test samples were evaluated by CYP450-carbonmonoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human CYP450 isoforms such as CYP3A4, CYP2D6, CYP2C9 and CYP1A2 isozymes were analyzed through fluorescence microplate screening assay. The combination index (CI) - isobologram method was applied for the prediction of combination effects. Plant extract showed significantly higher IC50 value than respective positive control against CYP3A4, 2D6, 2C9 and 1A2. Combination of the test extract and the phytoconstituent present therein along with oral hypoglycemic drugs also showed significantly less (P<0.001, P<0.01) interaction potential than known CYP450
2
inhibitors. Present study concluded that selected medicinal plant unlikely to cause significant herb-drug interactions and safe to use in management of diabetes.
List of abbreviations: CYP450
Cytochrome P450
HTS
High - throughput screening
IC50
The 50% inhibitory concentration
RP-HPLC
Reverse phase high-performance liquid chromatography
CYP450-CO
Cytochrome P450- carbon monoxide
RLM
Rat liver microsome
CI
Combination Index
Keywords: Herb-drug interaction, RP-HPLC, Berberis aristata DC., Cytochrome P450, Berberine, Combination index
1. Introduction Berberis aristata DC. (Family: Berberidaceae) is traditionally used in the Indian Systems of Medicines (ISM) for several ailments. The bark of this plant posses anti-diabetic, anti-inflammatory, hypotensive, immuno-stimulating, wound healing preparations, and also prevents infection of eyes [1, 2]. Plant extracts of Berberis aristata are widely used in the management of diabetes in therapies of the Indian system of medicine. Several studied revealed that Berberis aristata has an anti-diabetic activity through different mechanisms such as mimicking insulin, increasing insulin action by activating AMPK (5′ adenosine monophosphateactivated protein kinase), reducing insulin resistance through protein kinase
3
C-dependent up-regulation of insulin receptor expression, glycolysis induction, and by inhibiting DPP-IV activity [3]. Cytochrome P450 (CYP450) enzymes are major drug metabolizing enzymes and indicators of herb-drug interactions [4]. An inhibition or induction of CYP450 is the most common mechanism for the pharmacokinetic interactions of herb and drugs [5]. The safety issues, specifically herb–drug interaction data are important for the safe clinical practice of herbal medicines [6]. Multidrug combination therapy is common nowadays to achieve a synergistic therapeutic efficacy. Even though herbal medicines are gaining popularity as an alternative or adjuvant treatment globally, herb-drug and herb-herb interactions are a current topic of debate. Still effects of the herbal medicine in the combination of modern pharmaceuticals and possible adverse effects from herb - drug interactions remain to be verified. These studies are being suggested by most of the international regulatory bodies such as European Agency for Medicines (EMA) and US Food and Drug Administration (FDA) in the process of herbal drug development [7]. Herbal medicines having anti-diabetic activity are most commonly consumed as a dietary supplement with the prescribed medicines. Glimepiride and gliclazide are second generation sulphonylurea (oral hypoglycaemic drugs) most commonly used in the treatment of non-insulin-dependent diabetes mellitus [8]. The development of a marker profiling as a standardization parameter is of great importance for quality control management and batch to batch reproducibility of their therapeutic potency by the herbal drug manufacturers [9]. Based on these concepts the present study was designed to standardize the B. aristata extract with respect to berberine through reverse phase high-performance liquid chromatography (RPHPLC). The herb-drug interaction potential of these plant extracts, major phytoconstiuents in combination with oral hypoglycaemic drugs were investigated in order to obtain information about synergistic effects and about the safety profile in a combination therapy. Further we applied the combination index (CI)-isobologram method which is widely used to study
4
interaction to determine the nature of toxicological interactions of the B. aristata extract and oral hypoglycaemic drug, it allows computerized quantitation of synergism, additive effect and antagonism.
2. Materials and methods 2.1. Chemicals Vivid® CYP450 Screening Kit (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) and specific Vivid® Substrates were purchased from Invitrogen Drug Discovery Solutions (USA). Vivid® blue screening kit included baculosome (isozymes + NADPH-P450 reductase); regeneration system (glucose-6-phosphate + glucose-6-phosphate dehydrogenase) and NADP+ were used for the study. Ketoconazole and α-naphthoflavone were obtained from Merck (Mumbai, India). Quinidine and sulfaphenazole were procured from Sigma (Steinheim, Germany). Fluorimetric screening was performed using the 96 well black microplate (NUNC, Roskilde, Denmark; Catalogue no.: 137101). Berberine, glimepiride and gliclazide were purchased from Sigma Aldrich (Steinheim, Germany). Milli-Q Water (Millipore, USA) was used throughout the analysis; HPLC grade methanol, acetic acid, and other analytical grade solvents were procured from Merck (Mumbai, India). 0.45 μm syringe filter and membrane filter were obtained from Phenomenex (Torrance, CA) and Millipore (Billerica, MA) respectively. Milli-Q Water (Millipore, USA) was used throughout the analysis. All the chemical and reagents used were freshly prepared and analytical grade.
2.2. Plant material and extraction Bark of Berberis aristata was procured from the local vender and authenticated by Dr. S. Rajan, Field Botanist, Tamilnadu. Voucher specimen (SNPS-JU/1047) has been deposited at the School of Natural of Product Studies, Jadavpur University, Kolkata, for the future
5
reference. 200 gm collected plant material was extracted with 95% ethanol by cold maceration method. Solvent was removed by rotary vacuum evaporator (EYELA, Tokyo, Japan) in reduced pressure at 40°C, leaving semi-solid residue. The semisolid residue was dried under lyophilisation to get fine powder and yield of the extract was 12.38%.
2.3. Standardization of extracts through RP-HPLC The extract of B. aristata was standardized through RP-HPLC. The RP-HPLC (Waters 600, Milford, MA, USA) system was comprised of rheodyne-7725 injection valve with a sample loop (20 mL), vacuum degasser, quaternary pump and photo-diode array detector (PDA) with data acquisition by Empower TM 2 software. Different concentrations of the standard were prepared for the preparation of the calibration curve. Samples were filtered through 0.45 mm (NYL) syringe filter before injection. Retention time for all the eluting components were set within16 min run time. Chromatography was performed on a Spherisorb C18 column (250mm × 4.6 mm, 5 mm; Waters, Ireland) fitted with a C18 guard column (10 × 3.0 mm). The sample elution was performed at 25°C and detected at 254 nm. Calibration curve was constructed by the corresponding peak area and the concentration of standards by means of linear regression.
2.4. In vitro Cytochrome P450 inhibition studies 2.4.1. Preparation rat liver microsomes Rat liver microsomes were prepared based on the method described by Ponnusankar et al. (2011) [10]. In short, rats were sacrificed and the liver was isolated and treated with 1.15% potassium chloride solution (KCl) and then its size reduction was undertaken. 20% of the homogenized liver suspension was centrifuged through Beckman Coulter 64RALLEGRA at 9000 x g for 20 min. The clear supernatant was collected and subjected for re-
6
centrifugation at 105,000 x g 4 1C for 1 h (ultra centrifuge- SORVALL RC100). From the centrifuged suspension pellet microsomal fractions were collected and re-suspended in 1.15% KCl solution and kept at - 80 ºC for next use. Then estimation of protein was performed by a modified biuret method using bovine serum albumin (BSA) as standard by an AMS Photoanalyzer PF-2 (Roma, Italy). All experiments were performed as approved by the Institutional Animal Ethical Committee with the ethical guidelines as provided by the committee (approval number: AEC/PHARM/1501/05/2015) for the purpose of control and supervision of experiment on animals (CPCSEA), India.
2.4.2 Cytochrome P450-carbon monoxide (CYP-CO) complex assay CYP450-CO complex assay was performed with pooled rat liver microsomes (RLM) in 96 well microplates by the method described by Ponnusankar et al., (2011) [11]. In brief, the plant extract was mixed with ethanol and DMSO. It was incubated with RLM (liquefied with phosphoglycerol buffer; pH 7.4). The reaction between CYP450 and extract was initiated by adding an NADPH generating system (4.20 mg/mL of NADP + in solution of 100 mM glucose 6-phosphate, 100 mM MgCl2 and 100 U/mL glucose 6-phosphate dehydrogenase). The reaction between enzyme and extract was carried out in two microplates: one under room temperature (P) and another in carbon monoxide (CO) chamber (PC) for 15 min, while 0.5 M sodium hydrosulphite was used as the reducing agent. Two aliquots were placed in microplate wells and the other one was designed as reduced P450 (P) and other one was reduced P450-CO complex (PC). The P was sealed and kept outside the chamber whereas the PC well was kept in CO chamber and incubated for 15 min. All the samples were reduced by addition of 0.5 M sodium hydrosulfite (SHS) fresh solution. Visible appearance of PC sample was yellow while sample P remained colourless. The difference in absorbance of samples at 450 and 490 nm was monitored using BIORAD microplate reader
7
(Model 680XR). Ketoconazole was used as positive control and proper solvent controls were used for the study. Ethanol and DMSO were used as solvent for the cytochrome assay and had no interaction with spectral absorption while the solvent effect was neutralized by deionized water (Milli- Q water) and used as a solvent control. The result was expressed as percentage inhibition of CYP450. Concentration of CYP450 was calculated using the following formula [12]. [CYP450] (mM) = (ΔAPC-ΔAP)/91 Where, ΔAPC = absorbance difference of the PC sample and ΔAP = absorbance difference of the P sample. Percentage inhibition was calculated using the following formula: Percentage inhibition (PI) = (Blank - Test) × 100/Blank
2.4.3. Flurogenic high throughput screening (HTS) assay cytochrome P450 enzymes inhibition assays The assay was performed as per the method described by the Pandit et al., (2011a; 2012). In brief 145 μl NADPH-Co factor mixture (1.3 mM NADP+, 66mM MgCl2 and 66 mM glucose 6-phosphate) was added to each well of the first row of black 96 micro-well plates. Then 100 μl cofactor mixtures were added to the remaining wells except the blanks. 5 μl of extract solution was added to the first row of each well. Two-fold serial dilution was made and plates were incubated for 10 min at 37 °C. Quinidine (1 μM), ketoconazole (10 μM), sulfaphenazole (10 μM) and α-naphthoflavone (3 μM) were used as positive inhibitors for CYP2D6, CYP3A4, CYP2C9 and CYP1A2 respectively. 100 μl enzyme-substrate solutions were prepared in phosphate buffer and were used for starting the reaction and kept for 30 min. After incubation the reaction was stopped by addition of 0.5 M tris base. Product formation from the fluorogenic probes were determined from the fluorescence data at eight different concentrations of the inhibitors and the test. All measurements were performed in
8
triplicate. Calculation of IC50 values was based on the curves of mean enzyme activity versus inhibitor concentration [12]. The data were analyzed and result was expressed as IC50 value of the extract. Percentage inhibition = 100 – [(Signal of well-Blank)/ (Solvent control-Blank) × 100] IC50 = [{(50-LP) × (HC-LC) + LC}/ {HP-LP}] Where, LP = Low percentage of inhibition; HP = High percentage of inhibition; LC = Low concentration; HC = High concentration 2.5. Combination index (CI) - isobologram for determination of herb-drug interaction The herb drug interactions were investigated by flourescence microplate screening assays using 96-well micorplate readers. The IC-50 values were determined by different concentrations of each sample in triplicate. Combination index (CI) - isobologram equations was developed for the determination of the combined effect of herb-drug interaction. Two oral hypoglycaemic drugs were selected with the different combinations of plant extract and their active metabolite such as Berberis aristata + gliclazide, berberine +glimepiride, berberine + gliclazide and Berberis aristata + glimepiride for all the selected isozymes. The computer software CompuSyn [13] was used for the determination of the combination index (CI) to find out the combination effect. CI < 1, CI=1 and CI > 1 indicates synergism, concentration addition and antagonism respectively [14]. The CI method allows quantitative determination of synergism or antagonism and the elucidation of the mechanism of combined actions needs to be explored in other ways [15].
2.6. Statistical analysis Results were presented as Mean ± SEM. The results were subjected to one-way analysis of variance (ANOVA) followed by the Bonferroni test. The difference between the
9
means was considered significant when p<0.05 and above. The statistical analysis was performed using the GraphPad InStat Version 5 (USA).
3. Results 3.1. Standardization of extract through RP-HPLC The extract was standardized by RP-HPLC under isocratic conditions using the bexternal standard calibration technique. The Bioactive compound was identified by comparing with the respective retention time of the reference standard. Calibration curve was plotted by plotting peak areas against concentrations at five standard marker ranges from 100 – 500 μg/ml for barbarine with the flow rate of 1mL/min. Standard compound showed good linearity between concentrations and the peak area. The correlation coefficient (r2) of berberine was found to be 0.998. The chromatograms obtained from RP-HPLC analyses of the B. aristata extract and biomarkers have been shown in Fig.1. Optimum separation of berberine was achieved by using methanol: water with 1% acetic acid with 60:40 ratios (v/v). Retention time of berberine was found to be 14.28 min. Results were considered satisfactory and acceptable for subsequent quantitative analysis. The percentage of standard berberine was found to be 2.12% (w/w). 3.2. Cytochrome inhibition study 3.2.1. Cytochrome P450-CO complex assay CYP-CO assay was performed to evaluate the inhibitory potential of B. aristata and bioactive compounds in combination with glimepiride and/or gliclazide (oral hypoglycaemic drugs). The concentration of protein present in isolated rat liver microsomes was found to be 6.70 mg/ml. The CYP450 concentration of RLM was found to be 0.618 nmol/mg protein. The study was performed with different concentrations of the test solutions and the change in the absorption spectra was measured. The percentage inhibition of CYP450 was calculated and the comparison of percentage of inhibition of the individual test substances with respect 10
to positive control has been shown in Fig. 2. To ensure the possible inhibition by the extract, suitable solvent control was used in this experiment and the percentage inhibition was calculated after nullifying the solvent effect. Result indicated the concentration-dependent inhibition of CYP450. Positive inhibitor ketoconazole showed significantly higher (p<0.001) inhibition potential than extracts, bioactive, oral hypoglycaemic drugs and their combinations. Results showed the interaction of extracts with the pooled CYP450 was higher than compared to their designated bioactive molecule. This effect may be related to potentially synergistic activities of some other active molecules present in the extract [16, 17].
3.2.2. Fluorogenic HTS assays For the characterization of the herb - drug interactions potential, CYP isozymes specific high-throughput screening (HTS) assays is an important tool. The B. aristata extract and major bioactive principle berberine were evaluated for their capability to predict CYP450 mediated adverse drug effects of oral hypoglycaemic drugs such as glimepiride and gliclazide reaction when administered concomitantly. The interaction was studied with different CYP isozymes like CYP3A4, 2D6, 2C9 and 1A2 for their potential activity. The assay was based on the ability of a drug to compete with different fluorogenic substrates for different CYP isoforms. Coumarin derivatives were used as the probe substrates, which generated fluorescent products after dealkylation by CYPs. BOMCC (7-benzyloxymethyloxy- 3cyanocoumarin) was used as substrate for CYP3A4 and CYP2C9. In case of CYP2D6 and CYP1A2, EOMCC (7-ethoxymethoxy- 3-cyanocoumarin) was used as substrate. 7Hydroxycoumarin and 7-hydroxy-3-cyanocoumarin by products were formed against BOMCC and EOMCC after the enzymatic reaction. Positive controls for each isozyme were
11
used to confirm the assays precision. Testing of pure compounds indicated the overall activity explainable by tested compounds or caused by other constituents in the extract. Test samples showed concentration dependent inhibitory activity on CYP1A2, 2C9, 2D6 and 3A4 isozymes (Fig. 3). All the samples were assayed in triplicate and IC50 values were determined. IC50 values of the Berberis aristata extract, berberine and their combinations with oral hypoglycaemic drugs have been represented in Table 1. The results revealed that extract and its bioactive compound have less inhibition potential on the tested isozymes compared to their respective positive inhibitors. The study indicated that bioactive constituents have higher inhibition potential compared to extracts. Higher enzyme inhibition potential by the extracts may be due to the synergistic effects of other constituents present in the extract. More interestingly berberine + glimapiride and berberine + gliclazide combinations showed more inhibition potential against the isozymes studied. Berberine showed significantly less interaction potential against CYP450 isozymes compared to respective positive inhibitors. Test substances produced only minor inhibition of CYP1A2, 2C9, 2D6 and 3A4 isozymes. The cocktail of phyto-constituent and drugs showed significantly higher IC50 value compared to positive inhibitors against CYP1A2, 2C9, 2D6 and 3A4 isozymes. Results indicated that test substances have significantly less potential to interact with co-administered drug. 3.3. Combination index (CI) – isobologram for her-drug interaction The combination index (CI) method can be used for the prediction of combination action on all effect levels for multiple components and is widely used [18]. Herb - drug interaction of the Barberis aristata extract and their bioactive molecule berberine with the oral hypoglycaemic drugs glimepiride and gliclazide were evaluated. On the basis of observed CI values the binary mixture showed slightly synergetic effects of the oral hypoglycaemic drug with the Barberis aristata extract and their bioactive compound berberine (Table 2).
12
4. Discussion Development of marker profiling for standardization of herbal medicine is one of the most important parameter for maintaining their quality and to enrich the knowledge about the optimal concentrations of bio-active constituents present therein [19]. During herbal drug development the herb-drug interactions and nutritional supplement-drug interactions should not be ignored. Most of herbal medicines have been used without the prior consideration of physicians and there are chances of synergetic effects [20]. Two or more drugs administered together are considered to act synergistically if the observed effect is greater than the effect expected from the effect of each drug administered alone [21]. Considering the worldwide popularity of herbal medicine with the development of several herbal preparations, incidence of herb-drug interactions is predicted to be increased [22]. An approach has been made to evaluate the synergetic effects of anti-diabetic Indian medicinal plant B. aristata through CYP-CO complex method and high throughput fluorescence screening assays. Orally administered medicinal products are mostly metabolized by gut flora before being absorbed into the systematic circulation [23]. CYP450 has very significant role as metabolizing enzyme which is involved in the biotransformation of substances taken by oral route while the pre- systemic metabolism of these drugs takes place before entering into liver cell [17]. The interaction between herb and drug with CYP450 enzyme may alter plasma concentrations and may lead to toxic effects. Fluorescence based CYP450 screening method for the herbal medicine is important to obtain the knowledge about herb-drug interaction. Concentration dependent CYP450 inhibition was found in fluorescence screening assay by the test and standard [24]. CYP3A4, 2C9, 2D6 and 1A2 are clinically most important drug metabolizing enzymes. Although B. aristata showed little inhibition potential against CYP3A4 isozyme but with combination of oral hypoglycaemic drugs there was negligible inhibition. This effect may be due to the
13
glimepiride which is metabolized by CYP2C9 and has no effect with CYP3A4. Gliclazide mainly metabolized by CYP2C9 and 2C19 isozymes [8]. Results showed that B. aristata extract and the combination of oral hypoglycaemic drugs have not significant drug interaction potential against CYP2C9 & 3A4. In other word B. aristata and their bioactive compound did not involve in interaction with other drug metabolism. Combination index value showed that the B. aristata has little synergetic effects in the combination of the oral hypoglycaemic drugs. This may be due to the Barberis aristata and berberine also have hypoglycemic activity [25]. Further in-vivo and clinical study need to be performed in future for a better understanding of herb-drug interaction. 5. Conclusion With the increasing demand and popularization "herbs" and food as medicine across the globe, the quality and safety assessment of raw material becomes an essential parameter before marketing. The observed results by CYP-CO complex method and fluorogenic assay suggested that the B. aristata extract and their bioactive compounds in combination with oral hypoglycaemic agents, glimepiride and gliclazide have significantly less inhibitory effect on drug metabolising enzymes. Test substances were less likely to produce significant herb-drug interactions when consumed along with conventional therapeutics.
Conflict of interest Authors have no conflict of interest.
Acknowledgement Authors would like to express their gratitude to the Department of Biotechnology (DBT), Government of India, New Delhi, for providing TATA Innovation Fellowship to Dr. Pulok K. Mukherjee, School of Natural Product Studies, Jadavpur University, Kolkata. 14
References 1.
H.S. Yogesh, V.M. Chandrashekhar, H.R. Katti, S. Ganapaty, H.L. Raghavendra, G.K. Gowda, Anti-osteoporotic activity of aqueous-methanol extract of Berberis aristata in ovariectomized rats, J. Ethnopharmacol. 134 (2011) 334–338.
2.
D. Potdar, R.R. Hirwani, S. Dhulap, Phyto-chemical and pharmacological applications of Berberis aristata, Fitoterapia 83 (2012) 817–830.
3.
J. Singh, P. Kakkar, Antihyperglycemic and antioxidant effect of Berberis aristata root extract and its role in regulating carbohydrate metabolism in diabetic rats, J Ethnopharmacol 123 (2009) 2-6.
4.
H. Kim, H.K. Choi, T.C. Jeong, Y. Jahng, D.H. Kim, S. Lee, S. Lee, Selective inhibitory effects of mollugin on CYP1A2 in human liver microsomes, Food Chem. Toxicol. 51 (2013) 33-37.
5.
S. Pandit, P.K. Mukharjee, K. Mukharjee, R. Gajbhiye, M. Venkatesh, S. Ponnusankar, S. Bhadra, Cytochrome P450 inhibitory potential of selected Indian spices - possible food drug interaction, Food Res. Int. 45 (2012) 69–74.
6.
E.J. Choi, J.B. Park, K.D. Yoon, S.K. Bae, Evaluation of the in vitro/in vivo potential of five berries (bilberry, blueberry, cranberry, elderberry, and raspberry ketones) commonly used as herbal supplements to inhibit uridine diphospho-glucuronosyltransferase, Food Chem. Toxicol. 72 (2014) 13-19.
7.
B. Kamble, A. Gupta, I. Moothedath, L. Khatal, S. Janrao, A. Jadhav, B. Duraiswamy, Effects of Gymnema sylvestre extract on the pharmacokinetics and pharmacodynamics of glimepiride in streptozotocin induced diabetic rats, Chem. Bio. Inter. 245 (2016) 30-38.
15
8.
A. Tornio, M. Niemi, P.J. Neuvonen, T.B. Janne, Drug interactions with oral antidiabetic agents: pharmacokinetic mechanisms and clinical implications, Trends in Pharmacol. Sci. 33 (2012) 312-322.
9.
P.K. Mukherjee, S. Bahadur, S.K. Chaudhary, A. Kar, K. Mukherjee, Quality Related Safety Issue-Evidence-Based Validation of Herbal Medicine Farm to Pharma in P.K. Mukherjee (Edt) Evidence based validation of herbal medicine, Netherlands, USA 2015, 1-28.
10. S. Ponnusankar, S. Pandit, , R. Babu, A. Bandyopadhyay, P.K. Mukherjee, Cytochrome P450 inhibitory potential of Triphala- A Rasayana from Ayurveda. J. Ethnopharmacol. 133 (2011) 120-125. 11. S. Ponnusankar, S. Pandit, M. Venkatesh, A. Bandhopadhyay, P.K. Mukherjee, Cytochrome P450 inhibition assay for standardized extract of Terminalia chebula Retz. Phytother. Res. 25 (2011) 151- 154. 12. S. Pandit, S. Ponnusankar, A. Bandyopadhyay, S. Ota, P.K. Mukherjee, Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6, Phytother. Res. 25 (2011) 1429- 34. 13. T.C. Chou, N. Martin, 2005,CompuSyn for drug combinations: PC software and user's guide: A computer program for quantification of synergism and antagonism in drug combinations and the determination of IC50 and ED50 and LD50 values. ComboSyn, Inc., Paramus, NJ. 14. J. Fu, N. Zhang, J.H. Chou, H. Dong, S. Lin, G.S. Ulrich-Merzenich, T. Chou, Drug combination in vivo using combination index method: Taxotere and T607 against colon carcinoma HCT-116 xenograft tumor in nude mice, Synergy 3 (2016) 15-30. 15. I. Rodea-Palomares, A.L. Petre, K. Boltes, F. Legans, J.A. Perdigón-Melón, R. Rosal, F. Fernández-Piñas, Application of the combination index (CI) - isobologram equation to
16
study the toxicological interactions of lipid regulators in two aquatic bioluminescent organisms, Water Res. 44 (2010) 427-438. 16. S. Pandit, P.K. Mukherjee, S. Ponnusankar, M. Venkatesh, N. Srikanth, Metabolism mediated interaction of α-asarone and Acorus calamus with CYP3A4 and CYP2D6, Fitoterapia 82 (2011) 369-74. 17. R.K. Harwansh, K. Mukherjee, S. Bhadra, A. Kar, S. Bahadur, A. Mitra, P.K. Mukherjee, Cytochrome P450 inhibitory potential and RP-HPLC standardization of Trikatu - A Rasayana from Indian Ayurveda, J. Ethnopharmacol. 153 (2014) 674-681. 18. Y. Wang, C. Chen, Y. Qian, Z. Zhao, Q. Wang, X. Kong, Toxicity of mixtures of λcyhalothrin, imidacloprid and cadmium on the earthworm Eisenia fetida by combination index (CI) - isobologram method, Ecotoxicol Environment Safety. 111 (2015) 242-247. 19. P.J. Houghton and P.K. Mukherjee, Evaluation of herbal medicinal products, 1st ed. Pharmaceutical Press, London. 2009. 20. G.S. Ulrich-Merzenich, Combination screening of synthetic drugs and plant derived natural products-Potential and challenges for drug development, Synergy 1 (2014) 59-69. 21. N.J. Sucher, Searching for synergy in silico, in vitro and in vivo, Synergy 1 (2014) 3043. 22. S. J. Brantley, A.A. Argikar, Y. S. Lin, S. Nagar, M.F. Paine, Herb-drug interactions: Challenges and opportunities for improved predictions. Drug Metab. Dispos. 42 (2014) 301-317. 23. S.M. Ahmmed, P.K. Mukherjee, S. Bahadur, A. Kar, K. Mukherjee, S. Karmakar, A. Bandyopadhyay, Interaction potential of Trigonella foenum graceum through cytochrome P450 mediated inhibition. Ind. J. Pharmacol. 47 (2015) 530-534.
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24. P.K. Mukherjee, S. Ponnusankar, S. Pandit, P.K. Hazam, M. Ahmmad, K. Mukharjee, Botanicals as medicinal food and their effects on drug metabolizing enzymes, Food Chem. Toxicol. 49 (2011) 3142–3153. 25. Y. Wang, T. Campbell, B. Perry, C. Beaurepaire, L. Qin, Hypoglycemic and insulinsensitizing effects of berberine in high-fat diet- and streptozotocin - induced diabetic rats, Metabolism, 60 (2011), 298-305.
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Figure legends:
Fig.1: RP-HPLC chromatograms of berberine (A), B. aristata extract (B)
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Fig.2: Inhibitory effects of test extract, bioactive compounds and oral hypoglycaemic drugs and their combinations on pooled rat liver microsomes, (Values are expressed as mean ± SEM (n=3); **p<0.001 and ***p<0.01)
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Fig 3: Concentration dependant percentage inhibition of Berberis aristata extract, berberine, oral hypoglycaemic drugs and their combinations on drug metabolizing isozymes CYP3A4 (A), CYP2D6 (B), CYP2C9 (C) and CYP1A2 (D): (Values are in Mean ± SEM)
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Table 1: IC50 value (µg/ml) of extract and combination of extract and hypoglycemic drug: (One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test) Test samples
CYP3A4
CYP2D6
CYP2C9
CYP1A2
Berberis aristata
138.88±1.93
169.13±11.03
146.57±5.66
181.27±5.97
Berberis aristata + Glimepiride Berberis aristata + Gliclazide Berberine
81.88±2.14
110.94±2.60
98.32±2.04
131.16±4.12
96.65±2.27
105.01±1.22
83.95±1.57
125.44±2.75
65.80±3.63
85.60±2.57
79.85±5.23
98.64±5.70
Berberine + Glimepiride Berberine + Gliclazide
49.38±4.44
78.00±137
86.64±0.98
92.73±1.92
40.08±2.35
81.53±2.24
74.95±1.90
94.10±1.28
Glimepiride
56.82±5.93
40.48±5.21
46.32±5.79
79.59±3.29
Gliclazide
43.28±6.16
50.50±5.59
36.38±4.12
63.06±2.35
Positive control
Ketoconazole
Quinidine
Sulfaphenazole
α-naphthoflavone
0.05 ± 1.24
0.64 ± 0.94
0.09 ± 1.92
0.39 ± 0.58
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Table 2: Combination index (CI) values of Berberis aristata and berberine in the binary combinations with glimepiride and gliclazide
ED50
CI values ED75 ED90
ED95
CYP3A4 Berberis aristata + Glimepiride
0.88
0.79
0.71
0.66
Berberis aristata + Gliclazide
0.82
0.85
0.89
0.91
Berberine + Glimepiride
0.92
0.85
0.78
0.74
Berberine + Gliclazide
0.86
0.80
0.75
0.71
Berberis aristata + Glimepiride
0.80
0.71
0.77
0.87
Berberis aristata + Gliclazide
0.96
0.92
0.91
0.93
Berberine + Glimepiride
0.92
0.85
0.80
0.77
Berberine + Gliclazide
0.85
0.72
0.75
0.85
Berberis aristata + Glimepiride
0.83
0.58
0.59
0.65
Berberis aristata + Gliclazide
0.98
0.72
0.79
0.93
Berberine + Glimepiride
0.94
0.81
0.70
0.63
Berberine + Gliclazide
0.87
0.83
0.80
0.77
Berberis aristata + Glimepiride
0.69
0.66
0.82
1.00
Berberis aristata + Gliclazide
0.78
0.60
0.67
0.78
Berberine + Glimepiride
0.92
0.91
0.91
0.91
Berberine + Gliclazide
0.81
0.77
0.73
0.71
Test samples
CYP2D6
CYP2C9
CYP1A2
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